Method and probe set for detecting cancer

ABSTRACT

Methods for detecting cancer that include hybridizing a set of chromosomal probes to a biological sample obtained from a patient, and identifying if aneusomic cells are present in a selected subset of cells obtained from the biological sample are described. A set of chromosomal probes and kits for detecting cancer that include sets of chromosomal probes, are also described.

TECHNICAL FIELD

The invention relates to detecting cancer.

BACKGROUND OF THE INVENTION

Bladder cancer represents the fifth most common neoplasm and the twelfthleading cause of cancer death in the United States, where over 53,000new cases are diagnosed each year. Over 95% of bladder cancer cases inthe United States are transitional cell carcinoma (TCC, sometimesreferred to as urothelial cell carcinoma). Tumor stage is the bestpredictor of prognosis for patients with bladder cancer. Bladder canceris staged according to the depth of invasion of the tumor and whether ornot there are lymph node or distant metastases. Non-invasive papillarytumors (the most common and least aggressive type of bladder tumor) arereferred to as stage pTa tumors. “Flat” TCC, more commonly referred toas “carcinoma in situ” (CIS) is a more aggressive but less common tumorthat is associated with a high rate of progression to invasive disease.CIS is assigned a stage of pTIS. Tumors that have invaded through thebasement membrane of the epithelium into the underlying lamina propriaare assigned a stage of pT1. A tumor that has invaded the muscle of thebladder is a stage pT2 tumor. Invasion through the muscle into thetissue surrounding the bladder is a pT3 tumor. Invasion into surroundingorgans is a pT4 tumor. The term “superficial” bladder cancer refers topTa, pTIS, and pT1 tumors. Muscle-invasive bladder cancer refers to pT2,pT3, and pT4 tumors.

Approximately 80% of bladder cancer cases present as “superficial”bladder cancer and the remaining 20% as muscle-invasive bladder cancer.Patients with “superficial” bladder cancer do not require cystectomy(i.e. removal of the bladder) but have a high risk of tumor recurrence,and are monitored for tumor recurrence and/progression on a regularbasis (usually every 3 months for the first 2 years, every 6 months forthe next 2 years, and every year thereafter). Treatment for superficialbladder cancer generally consists of surgical removal of papillarytumors and treatment of CIS with Bacillus-Calmette Guerin (BCG).Patients with muscle invasive disease are treated by cystectomy and havea relatively poor prognosis compared to patients with “superficial”bladder cancer. Unfortunately, 80-90% of patients with muscle invasivebladder cancer initially present with muscle invasive disease. A largeshare of the estimated 10,000 deaths per year from bladder cancer isaccounted for by this group of patients. The fact that many patientswith advanced bladder cancer present that way suggests that screeningprograms that detect bladder cancer at earlier stages may help reducethe overall mortality from the disease. In fact, at least two largescreening studies suggest that screening does help identify bladdercancer at earlier stages. Messing et al., Urology, 45:387-396, 1995; andMayfield and Whelan, Br. J. Urol., 82(6):825-828, 1998.

Cystoscopy and urine cytology have been the mainstays for bladder cancerdetection over the past several decades. Several studies, however, haveshown that cytology has a disappointingly low sensitivity for bladdercancer detection. Mao et al., Science, 271:659-662, 1996; Ellis et al.,Urology, 50:882-887, 1997; and Landman et al., Urology, 52:398-402,1998. For this reason, there has been great interest in the developmentof new assays that have increased sensitivity for the detection ofbladder cancer. Examples of new assays that have been developed forbladder cancer detection include tests that detect bladder tumorantigens, e.g. BT test (C.R. Bard, Inc., Murrayhill, N.J.), NMP-22, FDP,etc., tests that detect increased telomerase activity (usuallyassociated with malignancy), or tests that detect genetic alterations inurinary cells and bladder washings (e.g. fluorescence in situhybridization (FISH) and microsatellite analysis). Although FISHanalysis may be more sensitive than other detection methods, largenumbers of cells must be counted, and consequently, the analysis is timeconsuming and costly. Therefore, a need exists for a rapid method ofdetecting cancer that maintains adequate sensitivity.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that a rapid,sensitive method for detecting cancer can be based on the presence ofaneusomic cells in a selected subset of cells from a biological sample.Selection of a subset of cells to be evaluated for chromosomal anomaliesreduces the number of cells to be analyzed, allowing analysis to beperformed in a rapid manner while maintaining, and even improving,sensitivity. The invention also provides a set of chromosomal probesselected to provide the optimal sensitivity in FISH analysis and kitsfor detecting cancer that include sets of chromosomal probes.

In one aspect, the invention features a method of screening for cancerin a subject. The method includes the steps of hybridizing a set ofchromosomal probes to a biological sample from the subject; selectingcells from the biological sample; determining the presence or absence ofaneusomic cells in the selected cells; and correlating the presence ofaneusomic cells in the selected cells with cancer in the subject. Thebiological sample can be urine, blood, cerebrospinal fluid, pleuralfluid, sputum, peritoneal fluid, bladder washings, oral washings, tissuesamples, touch preps, or fine-needle aspirates, and can be concentratedprior to use. Urine is a particularly useful biological sample. Thecells can be selected by nuclear morphology including nucleus size andshape. Nuclear morphology can be assessed by DAPI(4,6-diamidine-2-phenylindole dihydrochloride) staining. The method isuseful for detecting cancers such as bladder cancer, lung cancer, breastcancer, ovarian cancer, prostate cancer, colorectal cancer, renalcancer, and leukemia. The method is particularly suited for detectingbladder cancer.

The set of chromosomal probes includes at least three chromosomalprobes. The set can include at least one centromeric probe or at leastone locus specific probe. Suitable centromeric chromosomal probesinclude probes to chromosomes 3, 7, 8, 11, 15, 17, 18, and Y. A suitablelocus specific probe includes a probe to the 9p21 region of chromosome9. For example, the set can include centromeric chromosomal probes 3, 7,and 17, and further can include locus specific probe 9p21. Thechromosomal probes can be fluorescently labeled.

The invention also features sets of chromosomal probes and kits fordetecting cancer that include sets of chromosomal probes, that includecentromeric probes to chromosomes 3, 7, and 17, and further can includea locus-specific probe such as 9p21. The chromosomal probes can befluorescently labeled.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The invention advantageously provides a rapid, sensitive method fordetecting cancer, and can be used to screen subjects at risk for cancer,including solid tumors and leukemias, or to monitor patients diagnosedwith cancer for tumor recurrence. For example, subjects at risk forbladder cancer, lung cancer, breast cancer, ovarian cancer, prostatecancer, colorectal cancer, head and neck cancer, renal cancer, orleukemia can be screened or monitored for recurrence. In general, a setof chromosomal probes is hybridized to cells (from urine or otherbiological sample) on a slide. The cells on the slide are then visuallyscanned at a relatively low power (e.g. 200-400×) for morphologicfeatures strongly suggestive of malignancy (e.g. increased nuclear sizeor irregular nuclear shape). The nuclei of the cytologically abnormalcells are then examined for chromosomal abnormalities by switching theobjective to a higher power (e.g. 600-1000×) and “flipping” the filtersto determine if the cell is aneusomic or not. Use of this processmarkedly reduces the time spent assessing cells that have a lowprobability of being neoplastic and allows the examiner to focus theirefforts on the cells that have a much higher probability of beingneoplastic and showing aneusomy.

In Situ Hybridization

The presence or absence of aneusomic cells is determined by in situhybridization. “Aneusomic cells” are cells having an abnormal number ofchromosomes or having chromosomal structural alterations such ashemizygous or homozygous loss of a specific chromosomal region.Typically, aneusomic cells having one or more chromosomal gains, i.e.,three or more copies of any given chromosome, are considered testpositive in the methods described herein, although cells exhibitingmonosomy and nullisomy also may be considered test positive undercertain circumstances. In general, in situ hybridization includes thesteps of fixing a biological sample, hybridizing a chromosomal probe totarget DNA contained within the fixed biological sample, washing toremove non-specific binding, and detecting the hybridized probe.

A “biological sample” is a sample that contains cells or cellularmaterial. Typically, the biological sample is concentrated prior tohybridization to increase cell density. Non-limiting examples ofbiological samples include urine, blood, cerebrospinal fluid (CSF),pleural fluid, sputum, and peritoneal fluid, bladder washings,secretions (e.g. breast secretion), oral washings, tissue samples, touchpreps, or fine-needle aspirates. The type of biological sample that isused in the methods described herein depends on the type of cancer onewishes to detect. For example, urine and bladder washings provide usefulbiological samples for the detection of bladder cancer and to a lesserextent prostate or kidney cancer. Pleural fluid is useful for detectinglung cancer, mesothelioma or metastatic tumors (e.g. breast cancer), andblood is a useful biological sample for detecting leukemia. For tissuesamples, the tissue can be fixed and placed in paraffin for sectioning,or frozen and cut into thin sections.

Typically, cells are harvested from a biological sample using standardtechniques. For example, cells can be harvested by centrifuging abiological sample such as urine, and resuspending the pelleted cells.Typically, the cells are resuspended in phosphate-buffered saline (PBS).After centrifuging the cell suspension to obtain a cell pellet, thecells can be fixed, for example, in acid alcohol solutions, acid acetonesolutions, or aldehydes such as formaldehyde, paraformaldehyde, andglutaraldehyde. For example, a fixative containing methanol and glacialacetic acid in a 3:1 ratio, respectively, can be used as a fixative. Aneutral buffered formalin solution also can be used, and includesapproximately 1% to 10% of 37-40% formaldehyde in an aqueous solution ofsodium phosphate. Slides containing the cells can be prepared byremoving a majority of the fixative, leaving the concentrated cellssuspended in only a portion of the solution.

The cell suspension is applied to slides such that the cells do notoverlap on the slide. Cell density can be measured by a light or phasecontrast microscope. For example, cells harvested from a 20 to 100 mlurine sample typically are resuspended in a final volume of about 100 to200 μl of fixative. Three volumes of this suspension (usually 3, 10, and30 μl), are then dropped into 6 mm wells of a slide. The cellularity(i.e. density of cells) in these wells is then assessed with a phasecontrast microscope. If the well containing the greatest volume of cellsuspension does not have enough cells, the cell suspension isconcentrated and placed in another well.

Prior to in situ hybridization, chromosomal probes and chromosomal DNAcontained within the cell each are denatured. Denaturation typically isperformed by incubating in the presence of high pH, heat (e.g.,temperatures from about 70° C. to about 95° C.), organic solvents suchas formamide and tetraalkylammonium halides, or combinations thereof.For example, chromosomal DNA can be denatured by a combination oftemperatures above 70° C. (e.g., about 73° C.) and a denaturation buffercontaining 70% formamide and 2× SSC (0.3M sodium chloride and 0.03 Msodium citrate). Denaturation conditions typically are established suchthat cell morphology is preserved. Chromosomal probes can be denaturedby heat. For example, probes can be heated to about 73° C. for aboutfive minutes.

After removal of denaturing chemicals or conditions, probes are annealedto the chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Hybridization conditions vary, depending on theconcentrations, base compositions, complexities, and lengths of theprobes, as well as salt concentrations, temperatures, and length ofincubation. The higher the concentration of probe, the higher theprobability of forming a hybrid. For example, in situ hybridizations aretypically performed in hybridization buffer containing 1-2× SSC, 50%formamide and blocking DNA to suppress non-specific hybridization. Ingeneral, hybridization conditions, as described above, includetemperatures of about 25° C. to about 55° C., and incubation lengths ofabout 0.5 hours to about 96 hours. More particularly, hybridization canbe performed at about 32° C. to about 40° C. for about 2 to about 16hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash depend on the desired stringency. Forexample, for high stringency conditions, washes can be carried out atabout 65° C. to about 80° C., using 0.2× to about 2× SSC, and about 0.1%to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40).Stringency can be lowered by decreasing the temperature of the washes orby increasing the concentration of salt in the washes.

Chromosomal Probes

Suitable probes for in situ hybridization in accordance with theinvention hybridize (i.e., form a duplex) with repetitive DNA associatedwith the centromere of a chromosome. Centromeres of primate chromosomescontain a complex family of long tandem repeats of DNA, composed of amonomer repeat length of about 171 base pairs, that is referred to asalpha-satellite DNA. Non-limiting examples of centromeric chromosomalprobes include probes to chromosomes 3, 7, 8, 11, 15, 17, 18, and Y.Locus-specific probes that hybridize to a critical chromosomal region,such as the 9p21 region of chromosome 9, also are suitable.

Chromosomal probes are chosen for maximal sensitivity and specificity.Using a set of chromosomal probes (i.e., two or more probes) providesgreater sensitivity and specificity than use of any one chromosomalprobe. Thus, based on the results herein, chromosomal probes that detectthe most frequently aneusomic chromosomes, and that complement eachother, are included in a set. For example, based on discriminationvalues of probes determined herein, a set of chromosomal probes caninclude centromeric probes to chromosomes 3, 7, and 17. Additionally,the set can include probes to the 9p21 region of chromosome 9 or acentromeric probe to chromosome 8, chromosome 9, chromosome 11, orchromosome 18. As described herein, a probe to chromosome 7 when usedalone demonstrated a high sensitivity, and could detect about 76% ofbladder cancers. Probes to chromosomes 3 and 17, and to the 9p21 regionof chromosome 9 were able to detect additional bladder cancer cases thatshowed no abnormality with chromosome 7 probe alone. The combination ofprobes to chromosomes 3, 7, 17, and to 9p21 provide a sensitivity ofabout 95% for detecting bladder cancer in the cohort of patientsdescribed herein.

Chromosomal probes are typically about 50 to about 1×10⁵ nucleotides inlength. Longer probes typically comprise smaller fragments of about 100to about 500 nucleotides in length. Probes that hybridize withcentromeric DNA and locus-specific DNA are available commercially, forexample, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc.(Eugene, Oreg.), or from Cytocell (Oxfordshire, UK). Alternatively,probes can be made non-commercially from chromosomal or genomic DNAthrough standard techniques. For example, sources of DNA that can beused include genomic DNA, cloned DNA sequences, somatic cell hybridsthat contain one, or a part of one, human chromosome along with thenormal chromosome complement of the host, and chromosomes purified byflow cytometry or microdissection. The region of interest can beisolated through cloning, or by site-specific amplification via thepolymerase chain reaction (PCR). See, for example, Nath and Johnson,Biotechnic Histochem., 1998, 73(1):6-22, Wheeless et al., Cytometry,1994, 17:319-326, and U.S. Pat. No. 5,491,224.

Chromosomal probes typically are directly labeled with a fluorophore, anorganic molecule that fluorescesces after absorbing light of lowerwavelength/higher energy. The fluorophore allows the probe to bevisualized without a secondary detection molecule. After covalentlyattaching a fluorophore to a nucleotide, the nucleotide can be directlyincorporated into the probe with standard techniques such as nicktranslation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

Fluorophores of different colors are chosen such that each chromosomalprobe in the set can be distinctly visualized. For example, acombination of the following fluorophores may be used:7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™ (MolecularProbes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissaminerhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate(FITC), 7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and Cascade™blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). Probes areviewed with a fluorescence microscope and an appropriate filter for eachfluorophore, or by using dual or triple band-pass filter sets to observemultiple fluorophores. See, for example, U.S. Pat. No. 5,776,688.Alternatively, techniques such as flow cytometry can be used to examinethe hybridization pattern of the chromosomal probes.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as ³²P and ³H, although secondarydetection molecules or further processing then is required to visualizethe probes. For example, a probe indirectly labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard calorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Selection of Cells

According to the invention, cells are microscopically selected from thecells of a biological sample (e.g. urine, etc.) on a slide prior toassessing if aneusomic cells are present or absent. “Selecting” refersto the identification of cells that are more likely to be neoplastic dueto one or more cytologic (mainly nuclear) abnormalities such as nuclearenlargement, nuclear irregularity or abnormal nuclear staining (usuallya mottled staining pattern). These nuclear features, can be assessedwith nucleic acid stains or dyes such as propidium iodide or4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Propidium iodide isa red-fluorescing DNA-specific dye that can be observed at an emissionpeak wavelength of 614 nm. Typically, propidium iodide is used at aconcentration of about 0.4 μg/ml to about 5 μg/ml. DAPI, a bluefluorescing DNA-specific stain that can be observed at an emission peakwavelength of 452 nm, generally is used at a concentration ranging fromabout 125 ng/ml to about 1000 ng/ml. Staining of cells with DAPI orpropidium iodide is generally performed after in situ hybridization isperformed.

Determining Presence of Aneusomic Cells

After a cell is selected based on one or more of the stated criteria,the presence or absence of aneusomy is assessed by examining thehybridization pattern of the chromosomal probes (i.e. the number ofsignals for each probe) in each selected cell, and recording the numberof chromosome signals. This step is repeated until the hybridizationpattern has been assessed in at least 4 cells, if all 4 cells areaneusomic. In a typical assay, the hybridization pattern is assessed inabout 20 to about 25 selected cells.

Cells with more than two copies of multiple chromosomes (i.e., gains ofmultiple chromosomes) are considered cancer positive. Samples containingabout 20 selected cells and at least about 4 test positive cellstypically are considered cancer positive. If less than about 4 testpositive cells are found, the level of chromosome ploidy is determined.A cancer positive result also is indicated if more than 30% of the cellsdemonstrate hemizygous or homozygous loss (i.e., nullisomy) of aspecific chromosome region, such as loss of 9p21 in bladder cancer.Nullisomy can be confirmed as non-artifactual by observing thesurrounding normal appearing cells to see if they have two signals forthe specific chromosomal region.

Screening and Monitoring Patients for Cancer

The methods described herein can be used to screen patients for cancer,or can be used to monitor patients diagnosed with cancer. For example,in a screening mode, patients at risk for bladder cancer, such aspatients older than 50 who smoke, or patients chronically exposed toaromatic amines, are screened with the goal of earlier detection ofbladder cancer. The methods described herein can be used alone, or inconjunction with other tests, such as the hemoglobin dipstick test. Forexample, a patient having an increased risk of bladder cancer can bescreened for bladder cancer by detecting hemoglobin in the urine, i.e.,hematuria. During such a screening process, patients without hematuriado not need further analysis, and are instead, re-examined for hematuriain an appropriate amount of time, e.g., at their annual check-up.Samples from patients with hematuria are further analyzed using themethods described herein. In general, a set of chromosomal probes ishybridized with the biological sample, a subset of cells is selected,and the presence of aneusomic cells is determined in the selected cells.Patients that have aneusomic cells are further examined, for example, bycystoscopy, and can receive appropriate treatment, if necessary. Aftertreatment, patients are monitored for cancer recurrence using themethods described herein.

The superior sensitivity of the methods described herein indicate thatthis technique could replace cytology for the detection and monitoringof cancers such as bladder cancer. The majority of patients with bladdercancer will have detectable aneusomic cells and can be monitored fortreatment efficacy, and tumor recurrence/progression with the methodsdescribed herein. A small proportion of patients with cystoscopic orbiopsy evidence of bladder cancer (primarily those with low gradenon-invasive tumors) may not have detectable aneusomic cells in theirurine. These patients (i.e. those with low grade papillary tumors) areat very low rate of tumor progression and may be conveniently monitoredby a combination of the methods described herein and cystoscopy. Theappearance of anuesomic cells in the urine of these patients may heraldthe development of a more aggressive tumor in this subset of patients.The high sensitivity and specificity of the FISH test described hereinfor aggressive bladder cancers may help reduce the frequency ofcystoscopy.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1

Samples and Sample Preparation: Samples included voided urine from 21biopsy proven bladder cancer cases, in which a diagnosis was made byeither positive cytology or by histology, in the case of cytologynegative samples. Control urine samples included nine samples fromnormal healthy donors (age 25-80), and three samples from patients withgenitourinary diseases other than bladder cancer.

Approximately 50 to 200 ml of urine were collected per patient. Urinesamples were stored at 4° C. for less than 48 hours, and processed bycentrifugation at 1200 g for 5 minutes. The supernatant was discarded,and the pellet resuspended in 10 ml of 0.075 M KCl, and incubated atroom temperature for 15 minutes. Samples were spun for 5 minutes at 1200g, and the KCl solution was removed. Pellets were resuspended in 10 mlof a 3:1 methanol:glacial acetic acid fixative, and centrifuged for 8minutes at 1200 g. The fixative was carefully removed leaving the cellpellet, and this step was repeated two more times.

Density of the slides was monitored by frequently checking it under aphase contrast microscope, using a 20× objective, between droppings.Generally, it was attempted to obtain as many cells as possible on theslide without having any cell overlap. If a sample contained low numbersof cells, as much of the sample as possible was placed on the slide. Insamples with very low numbers of cells, the whole sample was used.Slides were dried overnight at room temperature.

Slides containing the samples were incubated in 2× SSC at 37° C. for10-30 minutes, then incubated in 0.2 mg/ml pepsin for 20 minutes at 37°C. Slides then were washed in PBS twice, for 2 minutes per wash, at roomtemperature. Cells were fixed in 2.5% Neutral Buffered Formalin for 5minutes at room temperature. Slides again were washed in PBS twice, for2 minutes per wash. After dehydration for 1 minute in each of 70%, 85%,and 100% ethanol, slides were used immediately, or stored at roomtemperature in the dark.

Three multicolor probe sets: A, B, and C were used in the initialhybridizations. Probe sets A-C contained the centromeric/locus specificprobes shown in Table 1. The color of the fluorophore used to label eachprobe also is shown in Table 1. Chromosomal probes (CEP®, chromosomalenumeration probe) were obtained from Vysis, Inc. (Downers Grove, Ill.).An aqua filter was used to visualize chromosomes 17 and 18. A yellowfilter was used to visualize the 9p21 locus specific probe, and a dualred/green filter or individual red or green filters were used tovisualize chromosomes 3, 7, 8, 9, 11, and Y.

TABLE 1 FISH Probe Sets Probe Spectrum Spectrum Spectrum Spectrum SetAqua Green Red Gold A 17 7  9 9p21 B 18 8 11 C 3 Y

Hybridization was performed by a HYBrite method or a conventionalmethod. In the HYBrite method, a HYBrite™ system from Vysis, DownersGrove, Ill., was used. Slides were placed on the HYBrite, and about 10μl of the probe set were added, covered, and sealed. The HYBrite wasprogrammed as follows: 73° C. for 5 min, then 37° C. for 16 hours.Slides then were washed in 0.4× SSC (0.06 M sodium chloride/0.006 Msodium citrate)/0.3% NP-40 for 2 minutes at 73° C., rinsed in 2×SSC/0.1% NP40 at room temperature, and air dried. Slides werecounterstained with approximately 10 μl of DAPI II (125 ng/ml of 4,6-diamidino-2-phenylindole dihydrochloride).

In the conventional method (i.e., Coplin jar method), a master mixcontaining chromosome probes was prepared in hybridization buffercontaining 50% formamide, 2× SSC, 0.5 μg/ml Cot1 DNA, and 2 μg/ml HPDNA. The probe mix was denatured at 73° C. for 5 minutes, and slideswere denatured in denaturation buffer (70% formamide, 2× SSC) in aCoplin jar at 73° C. for 5 min (6-8 slides/jar). Slides were rinsed ineach of 70%, 85%, and 100% ethanol for 1 minute. Approximately 10 μl ofhybridization mix were applied to each slide, covered with a coverslip,and sealed with rubber cement. Hybridization was performed in ahumidified chamber overnight at 37° C. Slides were washed in 0.4×SSC/0.3% NP-40 at 73° C. for 2 minutes, then rinsed in 2× SSC/0.1% NP-40briefly at room temperature. After air drying, slides werecounterstained with DAPI II. Samples were enumerated by recording thenumber of FISH signals in 100 consecutive cells.

In summary, this Example provides methods for isolating cells from abiological sample, and hybridizing a set of chromosomal probes to thecells.

Example 2

Detection of Bladder Cancer: Table 2 provides a summary of samplesanalyzed via cytology. Overall, urine cytology was positive in 13 out of21 cancer cases, equaling a sensitivity of 62%. This value is consistentwith published literature. Cytology detected tumor cells in 10 out of 11bladder cancer patients with advanced stages of the disease (first 11entries in Table 2). Among 10 patients with superficial bladder cancer(last 10 entries in Table 2), cytology was positive for 3 patients,equivocal for 3 patients, and negative for 4 patients. ND refers tonon-determined. Equivocal (E) refers to samples that were suspicious,but not diagnostic of transitional cell carcinoma (TCC). pTis refers tocarcinoma in situ, and pTa refers to non-invasive papillary carcinoma.pT1 refers to invasion of subepithelial connective tissue by the tumor;pT2 refers to invasion of muscle by the tumor; pT3 refers to invasion ofperivesical tissue by tumor; and pT4 refers to invasion of the prostate,uterus, vagina, pelvic wall, or abdominal wall.

The percent of aneusomic cells with four or more chromosome signalspermitted the greatest discrimination between the cancer group andnormal cases. Single copy gains or losses among the control group showedmuch higher frequencies and did not result in comparable sensitivitiesand specificities. The percentage of aneusomic cells, using thisdefinition, was determined for each group of samples (cancer and normal)and individually in each group. Using this definition, samples wereanalyzed in the following manner.

Discriminate analysis was performed to determine how well each probedifferentiated cancer cases from normal control, using the formula${{Discrimination}\quad {Value}} = \frac{\left( {{M1} - {M2}} \right)^{2}}{{SD1}^{2} + {SD2}^{2}}$

In this formula, M1 and M2 are the mean percent aneusomic cells withfour or more signals for cancer (n=21) and normal (n=9) groups,respectively, and SD1 and SD2 are standard deviations for each group ofsamples. Table 3 provides a summary of the results for the chromosomeenumeration probes (CEP) using this analysis.

TABLE 2 Detection of Bladder Cancer Via Cytology Patient Status StageGrade Cytology 215 Cancer ND ND positive 219 Cancer pT3 3 positive 224Cancer pTis 3 positive 228 Cancer ND ND ND 236 Cancer pTis 3 positive 66 Cancer pT3 3 positive D Cancer ND ND positive 229 Cancer pT3 3positive 171 Cancer pT4 3 positive 227 Cancer pT4 3 positive 240 CancerpT3 3 negative 191 Cancer pTa 2 positive 225 Cancer pTa 2 positive 230Cancer pTa 3 positive 223 Cancer pTa 2 E 234 Cancer pTa 2 E 235 CancerpTa 2 E 239 Cancer pTa 3 negative  69 Cancer pTa 2 negative 110 CancerpT1 3 negative  95 Cancer pT1 3 negative

TABLE 3 Probe Discrimination Values CANCER NORMAL Mean CEP 17 24.77 0.89SD (CEP 17) 25.44 1.76 Discrimination 0.88 Mean CEP 9 15.55 1.07 SD (CEP9) 19.33 2.01 Discrimination 0.55 Mean CEP 7 26.58 1.07 SD (CEP 7) 24.121.82 Discrimination 1.11 Mean CEP 18 13.09 1.63 SD (CEP 18) 14.35 1.80Discrimination 0.63 Mean CEP 11 16.32 1.52 SD (CEP 11) 19.44 1.87Discrimination 0.57 Mean CEP 8 15.58 1.52 SD (CEP 8) 15.47 1.87Discrimination 0.81 Mean CEP 3 27.91 0.49 SD (CEP 3) 26.33 0.78Discrimination 1.08

A discrimination value of >1.0, the 95% confidence interval around whichtwo populations are separated, was considered good (assuming normaldistribution and roughly equivalent standard deviation values). By thiscriteria, the best probes were 7, 3, and 17. This analysis providesinformation regarding the sensitivity and specificity of individualprobes but does not reveal the sensitivity/specificity of differentprobe combinations.

A cutoff of two standard deviations above the mean % of aneusomic cellsin normal cells was used as the criterion of FISH positivity forchromosomal gains. On the other hand, 9p21 changes in bladder cancer aremanifested as a loss and thus were evaluated as nullisomy. The cutoffvalue used for nullisomy was 3 standard deviations greater than the meanof normal samples. The percent aneusomic cells for each case by probeare shown in Table 4A for normal individuals and in Table 4B for cancerpatients. In Table 4B, samples below the double line are cytology falsenegative cases, and shaded text indicates false negative FISH resultsusing the cutoff values from Table 3. As shown in Table 4A, the percentof aneusomic cells (as defined by chromosomal gains) in normal samplesis low, ranging from about 0.5% to about 4.5%.

TABLE 4A Percent of aneusomic cells: normal cases CEP CEP CEP CEP CEPCEP CEP Patient 17 9 7 18 11 8 3 9p21 2F 0 0 0 0 0 0 0 0 3M 0 0 0 0 0 00 1.00 8M 0 0 0 0 0 0 0 0 9M 0 0 0 5 4 5 1 3.00 F5 4 2 2 2 1 1 0 ND F6 06 0 0 0 0 1.43 0 M12 4 1.67 6 3 5 4 2.00 14.00 M28 0 0 1.67 1.67 1.671.67 0 3.33 M23 0 1.07 0 3 2.00 2.00 0 15.00 MEAN 0.88 1.81 1.07 1.631.51 1.51 0.49 4.54 SD 1.76 2.01 1.81 1.79 1.87 1.87 0.77 6.29 Mean +4.41 5.09 4.7 5.2 5.26 5.26 2.05 17.12 2D

TABLE 4B Percent of aneusomic cells: cancer cases

TABLE 5 Results of FISH and Cytology for Normal and Bladder CancerPatients Bladder Cancer (n = 21) Non Cancer (n = 3) Cytology + Cytology− Cytology − FISH + 13 7 0 FISH −  0 1 3

Overall, as shown in Table 4B, chromosome 7 demonstrated the highestsensitivity of any individual probe (76%, 16/21). Probes to chromosome 3and 9p21 complemented chromosome 7 in that they were positive for someof the cases with normal chromosome 7 results. In combination, the threeprobes to chromosomes 3, 7 and 9p21 detected 20/21 bladder cancer casesand more importantly 7/8 cytology false negative cases (Table 4B). Inthis data set, this equates to an overall sensitivity of 95%.

The chromosome 17 probe (along with the chromosome 3 probe) detected thehighest number of cytology false-negative samples (4/8).

Example 3

Comparative Analysis with Standard Screening Methods: 190 patients fromthe Mayo clinic were prospectively enrolled in this study. A majority ofthe patients either had a previous diagnosis of bladder cancer or werebeing evaluated for a possible initial diagnosis of bladder cancer (e.g.for microhematuria). A small proportion of the patients were beingevaluated for genitourinary disorders other than bladder cancer. Testmethodologies compared included urine cytology, cystoscopy, BTA STAT(C.R. Bard, Inc., Murray Hill, N.J.), FISH, and hemoglobin dipstick(Bayer Corporation, Diagnostic Division, Elkhart, Ind.).

FISH generally was performed as described in Example 1. Approximately 25to 200 ml of urine were collected per patient. Urine samples were storedat 4° C. for up to 48 hours, and processed by centrifugation at 600 gfor 10 minutes. The supernatant was discarded, and the pellet wasresuspended in 25-50 ml of 1× phosphate-buffered saline (PBS). Aftercentrifugation for 5 minutes at 600 g, the supernatant again wasdiscarded. Pellets were resuspended slowly in 1.5-5 ml of fixative(methanol:glacial acetic acid, 3:1), and centrifuged for 5 minutes at600 g. The fixative was carefully removed, and this step was repeatedtwo more times.

After the final centrifugation, the fixative was removed, leaving anappropriate amount of the solution for dropping onto slides such as the12-Circle 6 mm Shandon Lipshaw slides. If the cell pellet was verysmall, and hardly visible to the eye, approximately 100 μl were leftabove the pellet. If the cell pellet was easily visible by eye, as muchCarnoy's fixative as possible was removed, and 0.5 ml of fresh Carnoy'sfixative was added. If no pellet was visible, the entire sample oftenwas dropped on one slide. Approximately 3, 10, and 30 μl of the cellsuspension were then dropped into three separate 0.6 mm wells of aShandon 12 well slide. The cellularity (i.e. density of cells) in thesewells was assessed with a phase contrast microscope. If the wellcontaining the smallest volume of cell suspension is too dense, the cellsuspension is further diluted and a portion of this dilution is put in afourth well. If the well containing the greatest volume of cellsuspension does not have enough cells, the cell suspension isconcentrated and placed in a fourth well.

Slides containing the samples were incubated in 2× SSC at 37° C. for10-30 minutes, then incubated in 0.2 mg/ml pepsin for 10 minutes at 37°C. Slides then were washed in PBS twice, for 2 minutes per wash, at roomtemperature. Cells were fixed in 2.5% Neutral Buffered Formalin for 5minutes at room temperature. Slides again were washed in PBS for 5minutes. After dehydration for 1 minute in each of 70%, 85%, and 100%ethanol, slides were dried on a slide warmer at 45-50° C. for 2 minutes.Slides were either left in 100% ethanol at 4° C. until further use, orwere used for FISH.

Hybridization was performed by a HYBrite method or a conventionalmethod. In the HYBrite method, a HYBrite™ system from Vysis, DownersGrove, Ill., was used. Slides were placed on the HYBrite, and about 3 μlof probe were added per target, then the slides were covered and sealed.The HYBrite was programmed as follows: 73° C. for 5 min, then 37° C. for16 hours. Slides then were washed in 0.4 SSC (0.06 M sodiumchloride/0.006 M sodium citrate)/0.3% NP-40 for 2 minutes at 73° C.,rinsed in 2× SSC/0.1% NP40 at room temperature, and air dried. Slideswere counterstained with approximately 3 μl of DAPI II (125 ng/ml of 4,6-diamidino-2-phenylindole dihydrochloride).

In the conventional method, a master mix containing chromosome probeswas prepared in hybridization buffer containing 50% formamide, 2× SSC,0.5 μg/ml Cot1 DNA, and 2 μg/ml HP DNA. The probe mix was denatured at73° C. for 5 minutes, and slides were denatured in denaturation buffer(70% formamide, 2× SSC) in a Coplin jar at 73° C. for 5 min (6-8slides/jar). Slides were rinsed in each of 70%, 85%, and 100% ethanolfor 1 minute. Samples were dried on a slide warmer for 2 minutes orless. Approximately 3 μl of hybridization mix were applied per target oneach slide, and the slides were covered immediately with a coverslip,which then was sealed with rubber cement. Hybridization was performed ina humidified chamber overnight at 37° C. Slides were washed in 0.4×SSC/0.3% NP-40 at 73° C. for 2 minutes, then rinsed in 2× SSC/0.1% NP-40briefly at room temperature. After air drying, slides werecounterstained with DAPI II. Twenty cytologically abnormal cells wereselected in each sample, and analyzed for their FISH pattern.

For 53 cancer cases, as diagnosed by biopsy (Stage/Grade) (n=49) andcytology positive TCC (n=34), cytology had a sensitivity of 57%(equivocal cytology diagnoses counted as positive), cystoscopy had asensitivity of 88% (equivocal cystoscopy results counted as positive),BTA STAT had a sensitivity of 71%, and FISH had a sensitivity of 86%. In63 cancer negative cases, as indicated by a negative cystoscopy with orwithout negative cytology and no history of bladder cancer, BTA STAT hada specificity of 73%, and FISH had a specificity of 90% (specificity ofcystoscopy and cytology cannot be determined since they were used todefine which patients did not have bladder cancer.

Two of the three patients with “false positive” FISH results also hadpositive telomerase, hemoglobin dipstick and BTA-STAT or BTA-TRAKresults. Should these two patients prove to have bladder cancer, thespecificity of FISH will approach 100%. This has implications for thepositive predictive value of the test as discussed below. Table 6 showsthe sensitivity of the various tests by stage and grade. In Table 6, “E”refers to equivocal, and “*” indicates 3 FISH⁺ samples were cancer bycytology only and were not included.

The positive and negative predictive values (PV), based on a diseaseprevalence of 28% (53/190), specificity of 93% and sensitivity of 77%,are indicated in Table 7.

TABLE 6 Grade 1 Grade 2 Grade 3 Overall Cytology pTa 1 + [1E]/8  5/132/2 39% (9/23)  pT1-pT4 3 + [3E]/9  66% (6/9)  pTis 5 + [5E]/12 83%(10/12) Overall 25% (2/8) 38% (5/13) 78% (18/23) 57% (25/44) CystoscopypTa 5 + [1E]/7 13/13 2/2 95% (21/22) pT1-pT4 4 + [3E]/9  78% (7/9)  pTis5 + [5E]/12 83% (10/12) Overall 86% (6/7) 100% 83% (19/23) 88% (38/43)(13/13) FISH pTa 4/7  9/12 2/2 71% (15/21) pT1-pT4 12/12 100% (12/12) pTis 10/10 100% (10/10)  Overall 57% (4/7) 75% (9/12) 100% (24/24)  86%(37/43) BTA STAT pTa 4/8  6/14 2/2 50% (12/24) pT1-pT4  9/10 90% (9/10) pTis 12/12 100% (12/12)  Overall 50% (4/8) 43% (6/14) 96% (24/25) 71%(33/46)

TABLE 7 Predictive Value Method PV + PV − FISH 77% 94% BTA STAT 51% 87%

Fifty-one out of the 53 cancer cases had a dipstick result. Thesensitivity of the hemoglobin dipstick test was 91% for stages pT1-pT4and 100% for carcinoma in situ (Tis), making it an ideal screening test(Table 8). However, the specificity (52%) of the hemoglobin dipsticktest was poor. While the high sensitivity and low cost of the hemoglobindipstick make it a useful screening test, it is clear that positiveresults must be confirmed by a more specific test. Cytology cannot bethe test of choice because it detected as positive only 33% of pT2-pT4cases and missed 2 of 12 carcinoma in situ (see Table 6).

TABLE 8 Hb dipstick Trace to 3 + Negative Sensitivity pTa 13 11 54%pT1-pT4 10  1 91% pTis 12  0 100% 

The clinical utility of FISH was compared to the standardcytology/cystoscopy testing regimen. Of the 53 cancer cases, there were22 cases of advanced stage disease (pT2-pT4 or pTIS). Failure to detectthese cases represents the most serious false negative situation. Whilecytology/cystoscopy was positive in 13/22 cases for a sensitivity of59%, FISH had 100% sensitivity, i.e., was able to detect 22/22 cases(see Table 9 for a representation of 5 of these cases). This indicatesthat FISH is an improved cytology test which has high sensitivity fordetecting advance stages of disease.

TABLE 9 Patient Stage Grade Cystoscopy Cytology FISH  79 pT2 Grade 3 E EPos 132 pT2 Grade 3 Neg Neg Pos  1 pT3 Grade 3 Neg Neg Pos 115 pT4 Grade3 E E Pos 180 pTIS Grade 3 E Neg Pos

Other Embodiments

It is understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of screening for bladder cancer in asubject comprising: (a) hybridizing a set of chromosomal probes to abiological sample from said subject, wherein said set comprises at leastthree chromosomal probes selected from the group consisting of probes tochromosomes 3, 7, 8, 11, 15, 17, 18 and Y; (b) selecting cells from saidbiological sample based on abnormalities in nuclear morphology, nuclearsize, or nuclear shape; and (c) determining the presence or absence ofaneusomic cells in said selected cells by examining the hybridizationpattern of said set of chromosomal probes in each of said selectedcells, wherein the presence of aneusomic cells in said selected cells isindicative of bladder cancer in said subject.
 2. The method of claim 1,wherein said biological sample is selected from the group consisting ofurine, blood, bladder washings, tissue samples, and touch preps.
 3. Themethod of claim 1, wherein said biological sample is concentrated. 4.The method of claim 1, wherein said biological sample is urine.
 5. Themethod of claim 1, wherein said chromosomal probes are fluorescentlylabeled.
 6. The method of claim 1, wherein said set comprises at leastone centromeric probe.
 7. The method of claim 6, wherein said setfurther comprises at least one locus specific probe.
 8. The method ofclaim 7, wherein said locus specific probe is to the locus 9p21.
 9. Themethod of claim 1, wherein said set comprises probes to chromosomes 3,7, and
 17. 10. The method of claim 9, wherein said set further comprisesa locus specific probe to the locus 9p21.
 11. The method of claim 1,wherein cells are selected by nuclear morphology.
 12. The method ofclaim 1, wherein cells are selected by nuclear size.
 13. The method ofclaim 1, wherein cells are selected by shape of nucleus.
 14. The methodof claim 11, wherein nuclear morphology is assessed by DAPI staining.15. A set of chromosomal probes, said set comprising centromeric probesto chromosomes 3, 7, and
 17. 16. The set of chromosomal probes of claim15, said set further comprising a locus-specific probe.
 17. The set ofchromosomal probes of claim 16, wherein said locus-specific probe is tothe locus 9p21.
 18. A kit for detecting cancer, said kit comprising aset of chromosomal probes, wherein said set comprises centromeric probesto chromosomes 3, 7, and
 17. 19. The kit of claim 18, said kit furthercomprising a locus-specific probe.
 20. The kit of claim 19, wherein saidlocus-specific probe is to the locus 9p21.
 21. The kit of claim 18,wherein said chromosomal probes are fluorescently labeled.
 22. The setof chromosomal probes of claim 15, wherein said chromosomal probes arefluorescently labeled.
 23. A kit for detecting cancer, said kitcomprising a set of chromosomal probes, wherein said set consistsessentially of centromeric probes to chromosomes 3, 7, and
 17. 24. Thekit of claim 23, wherein said set further comprises a locus specificprobe to 9p21.
 25. A set of chromosomal probes, said set consistingessentially of centromeric probes to chromosomes 3, 7 and
 17. 26. Theset of chromosomal probes of claim 25, said set further comprising alocus specific probe to 9p21.